Eastern equine encephalitis virus (EEEV) is one of the most highly pathogenic viruses in humans with 35-50% mortality and is an actively reemerging public health threat. EEEV is a New World alphavirus, a group of mosquito-borne encephalitic pathogens with a wide geographical distribution and broad host range that includes the related agents Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV). A better understanding of the viral and host determinants of New World alphaviral entry into host cells will lend insight into critical aspects of the viral life cycle and pathogenicity and may provide a basis for the development of future treatment strategies and therapies. A molecular understanding of New World alphaviral tropism and entry has been limited by the requirement for special biosafety containment facilities. To overcome these limitations, we have developed pseudotyped viruses, which contain the EEEV, WEEV or VEEV envelope glycoproteins on their surface and express a reporter gene as a marker of infection. Each pseudotyped virus recapitulates the native entry mechanisms of the specific viruses on which they are based and can be used in standard BSL-2 conditions. We have also generated soluble New World alphaviral E2 glycoprotein ectodomains that bind permissive target cells. These reagents enable a comparative investigation into the molecular details that govern the viral entry pathways of representative and important New World alphaviruses. The goals of this proposal are to define the receptor binding domains within the New World alphaviral envelope glycoproteins, to determine if New World alphaviruses share a common cell surface receptor and to identify their specific host entry factors. In the first aim, the candidate will generate soluble forms of New World alphaviral E2 glycoproteins with iterative N and C-terminal truncations and site-directed mutations and characterize their ability to bind target cells. In the second aim, the candidate will comparatively characterize New World alphaviral tropism on a broad range of target cells, map common receptor usage through a series of binding and infection competitions, and identify specific cell surface receptors by immunoprecipitation with soluble alphaviral E2 receptor binding domains. Through the support of the Broad Institute, in the third aim the candidate will identify specific host cell entry factors by high-throughput genetic screening of human cells that have become non-permissive to New World alphaviral pseudotype infection using a shRNA library. The importance of potential host factors for New World alphaviruses identified with soluble receptor binding domains or using the genetic screen with pseudotyped viruses will be confirmed in experiments with live virus at the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE BEID) BSL-3 core facility. These studies will be conducted under the mentorship of Dr. Michael Farzan and Dr. Hyeryun Choe who have a long-standing expertise in viral entry and are pioneers in the identification of viral receptors. The strategies in this proposal are closely modeled after their approach towards the identification of the receptors for the SARS coronavirus and hemorrhagic New World arenaviruses and the chemokine co-receptors for HIV. In addition to these proposed research aims and experiments, the candidate's career development goals are to gain expertise in basic virology, viral pathogenesis, protein structure-function, high throughput genetic screens and the ethical conduct of research. The candidate will achieve these immediate goals by attending a series of formal seminars and nanocourses in virology, protein biochemistry, genetic screening strategies and the responsible conduct of research. The candidate will also receive the necessary BSL-3 biocontainment, biosafety, and security training through NERCE BEID to perform confirmatory experiments with live New World alphaviruses at their core facility. The candidate's long-term career goals are to attain a tenure-track faculty position and to continue his research on the mechanisms of New World alphaviral entry and pathogenesis. The candidate's immediate research training goals and long-term career goals will also be facilitated through the guidance and mentorship of his scholarship oversight committee composed of a distinguished group of scientists with leading-edge expertise pertinent to the candidate's field of study. The training opportunities and resources at Children's Hospital Boston and Harvard Medical School together with the mentorship of Drs. Farzan and Choe are an ideal environment for his career development program. Children's Hospital Boston is committed to this career development plan and has assured the candidate will be able to devote at least 85% full-time effort to the activities described in this proposal.